Mesenchymal stem cells (MSCs) are considered one of the most promising tools for cell and cell-mediated gene therapy in skeletal repair. MSCs were shown to have the potential to differentiate into several lineages including bone (Haynesworth et al., 1992), cartilage (Mackay et al., 1998, Yoo et al., 1998), fat (Pittenger et al., 1996), tendon (Young et al., 1998), muscle, and stroma (Reviewed by Caplan et al, 2001). The main source known of MSCs in adult humans is the bone marrow compartment that contains several cell types including cells of the hematopoietic lineage, endothelial cells, and mesenchymal stem cells, which are part of the marrow stromal system (Pittenger et al, 1999).
Several protocols were recently established in order to enable regeneration and filling of large bone defects, utilizing human MSCs expanded in culture as both the cells differentiating into osteogenic cells, and as the vehicles delivering the therapeutic gene product such as BMP-2 (Turgeman et al., 2001). It was recently shown that in combination with BMP-2, hMSCs are able to heal full-thickness non-union bone defects (Turgeman et al., 2001), and in recent studies it was shown that human MSCs can be transduced by retroviral vectors and maintain stable expression of the therapeutic gene after in vivo transplantation (Lee et al., 2001). Within these studies, the MSCs were isolated from the bone marrow, expanded in culture, in some cases genetically engineered, and transplanted in-vivo. The culture expansion stage is extremely costly, time consuming, and in many cases, the cells may lose their multipotentiality and fail to achieve the desired goal. In contrast, very few studies described the use of non-cultured freshly isolated human MSCs. Horwitz et al., (1999) showed that human MSCs present in unprocessed bone marrow allografts engraft and may provide stem cell reservoir for osteoblast differentiation and renewal. The isolation of hMSCs-enriched population requires efficient and reproductive isolation method. Few methods were described for the isolation of MSCs, including enhancement of the plastic adherence property of the cells by using selected lots of fetal calf serum (Kadiyala et al., 1997, Pittenger et al., 1999), immunomagnetic isolation based on the presence of the STRO-1 surface molecule (Gronthos and Simmons, 1995). Both methods are very hard to be reproduced by other labs and no studies were done to show the differentiation potential of cells before culture expansion. Majumdar et al. (2000) showed that cells from human BM aspirates were isolated by the anti-CD105 (endoglin) antibodies, differentiated to chondrogenic cells after culture expansion, and showed immunophenotype distinctive to MSCs, suggesting that these CD105+cells contain the osteogenic MSCs population.